Deficiencies in DNA repair (mismatch repair deficiencies in colon cancers, nucleotide excision repair in melanomas), deficiencies in cell cycle checkpoints (Rb, p53, BRCA, and Chk2 deficiencies in solid tumors), and apoptosis (APC mutations in colon cancers, Bcr-Abl recombinations in leukemia, Bcl-2 overexpression in lymphomas) promote cancers. They also contribute to therapeutic responses and resistance to chemotherapy by preventing a normal apoptotic response of tumor cells bearing the above mutations. Our studies in DNA repair include studies with Ecteinascidin 743 (Et743) (NSC 648766), DNA alkylating agents, and topoisomerase inhibitors. Et743 is a novel anticancer agent in Phase II/III clinical trials. This drug is remarkable because of its clinical activity and its unique mechanism of action. Responses have been observed in sarcomas, which are notoriously resistant to other known treatments, as well as in ovarian and breast cancer. Et743 differs from other clinically used anticancer agents because it forms covalent adducts at specific guanines in the DNA minor groove and because it selectively Et743 traps the transcription-coupled NER (TC-NER) machinery to induce lethal DNA strand breaks. Thus, Et743 defines a novel class of anticancer drugs in which enhanced antiproliferative activity parallels enhanced cellular DNA-repair capability. These findings led us to study the NER-dependence for cisplatin. We found that defective TC-NER sensitizes cells to cisplatin, whereas defective global genome repair (GG-NER) did not affect cisplatin response. The complementary between the activities of Et743 and cisplatin with respect to TC-NER suggests the use of Et743 in cisplatin-resistant tumors and vice-versa. A clinical protocol has been proposed for a Phase I clinical trial of Et743 in ovarian cancers resistant to cisplatin (Collaboration with Dr. Elise Kohn, Pathology Branch, CCR, NCI). Further molecular studies are planned to determine the transcription- and the strand-specific-dependence of the DNA single-strand breaks induced by Et743. We are also looking at TC-NER-dependent transcription inhibition by microarray analyses using NER-deficient, XPD, and XPD-complemented cells. Because most cancers have alterations in the cell cycle checkpoint pathways (p53, pRb) and cell cycle machinery (cyclins, cyclin-dependent kinase inhibitors - such as p16), we are exploring inhibitors of cell cycle checkpoints as novel anticancer agents. 7-hydroxystaurosporine (UCN-01) is a novel anticancer agent in phase II/III clinical trials. We found that UCN-01 is synergistic with DNA damaging agents such as topoisomerase inhibitors and drugs that act during the S-phase of the cell cycle. This synergism has been related to an abrogation of the S-phase checkpoint, which is controlled by 2 protein kinases, Chk1 and Chk2. We found that UCN-01 inhibits both Chk1 and Chk2, and we are investigating the role of Chk2 in cell cycle checkpoint response in cancer cells. We have expressed Chk2 as a recombinant protein and preliminary experiments are ongoing to discover Chk2 inhibitors using a high throughput screen (collaboration with Drs. Shoemaker and Scudiero, DTP, NCI). Our studies on apoptosis led to the implication of APE-1 (APEX) in apoptotic DNA fragmentation. We had previously reported the presence of an unidentified apoptotic nuclease (AN34) in human leukemia HL60 cells. After purification and peptide sequencing, we found that AN34 corresponds to human apurinic endonuclease (APE-1 = APEX = REF-1). This identification has been confirmed by immunoblotting and immunoblocking experiments. Biochemical assays demonstrated that caspase 3 can activate the endo- and 3'-exo-nuclease of APEX during apoptosis. We are also studying the involvement of topoisomerase I in apoptotic DNA fragmentation.